Agent for improving ocular subjective symptoms and method thereof

ABSTRACT

Provided is an agent for proving human ocular subjective symptoms containing hyaluronic acid having an aminoalkyl cinnamate covalently bonded thereto.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/539,771, filed on Jun. 26, 2017, which was a 371 of InternationalPatent Application No. PCT/JP2015/086233, filed on Dec. 25, 2015, andclaims priority to Japanese Patent Application No. 2014-263710, filed onDec. 26, 2014.

TECHNICAL FIELD

The present invention relates to an agent for improving human ocularsubjective symptoms and method thereof.

BACKGROUND ART

Improvement of ophthalmic abnormalities has conventionally been assessedfocusing primarily on external observation and evaluation (objectivesign), while little emphasis has been placed on evaluation from thesubjective viewpoint of the patient in the manner of ophthalmicdiscomfort or distress (subjective symptoms). For example, theameliorative effects of dry eye, one of the ophthalmic abnormalities,have come to be evaluated by focusing primarily on objective sign suchas normalization of tear fluid volume or decreases in the damaged areasof corneal and conjunctival epithelia. In actuality, although sodiumhyaluronate, diquafosol sodium and rebamipide in Japan, and cyclosporinein the U.S., have been approved for use as ophthalmic solutionsprescribed for dry eye, ophthalmic solutions have yet to be approvedthat satisfy the requirements of improving subjective symptoms inaddition to improving objective sign.

Although hyaluronic acid having a hydrophobic residue bound thereto isknown to be effective against ophthalmic abnormalities such as dry eye(see, for example, Patent Document 1), the efficacy thereof isessentially based entirely on “objective sign” as described above, andit has yet to be evaluated from the viewpoints of improvement and/ortreatment of subjective symptoms.

PRIOR ART DOCUMENTS Patent Documents

Patent Document 1: JP-T 2009-511423

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Objective sign and subjective symptoms do not necessarily indicatesimilar tendencies, and subjective symptoms may be poor while objectivesign are favorable, or conversely, objective sign may be poor whilesubjective symptoms are favorable. In addition, existing ophthalmicsolutions prescribed for dry eye require the patient to administer theophthalmic solution over a long period of time until the efficacythereof is demonstrated, resulting in the problem of patients withserious symptoms of dry eye losing the will and desire to continue topatiently administer the same ophthalmic solution over a long period oftime, and thereby creating a desire for the providing of a drug capableof demonstrating its effect immediately after the start ofadministration.

An object of the present invention is to provide an agent and method forimproving human ocular subjective symptoms and method thereof.

Means for Solving the Problems

As a result of conducting extensive studies to solve the aforementionedproblems, the inventors of the present invention found that a compoundin which an aminoalkyl cinnamate is covalently bonded to hyaluronic acid(hereinafter referred to as “HA”) demonstrates the effect of improvingsubjective symptoms of the eyes in humans, thereby leading to completionof the present invention.

Namely, the present invention provides an agent for improving humanocular subjective symptoms (hereinafter referred to as the“pharmaceutical preparation of the present invention”) containing HAhaving an aminoalkyl cinnamate covalently bonded thereto. Thispreparation is in the form of a solution and the HA having an aminoalkylcinnamate covalently bonded thereto is preferably contained at aconcentration of 0.3% (w/v). In addition, the human to whom thispreparation is administered is preferably a person with dry eye.

In addition, the present invention provides a method for improving humanocular subjective symptoms that comprises a step for administering HAhaving an aminopropyl cinnamate covalently bonded thereto to a human eye(hereinafter referred to as the “method of the present invention”). ThisHA having an aminopropyl cinnamate covalently bonded thereto ispreferably administered in the form of a solution having a concentrationof 0.3% (w/v). In addition, the human to whom this solution isadministered is preferably a person with dry eye.

Effects of the Invention

According to the present invention, an HA-based preparation forimproving human ocular subjective symptoms and a method for improvinghuman ocular subjective symptoms using that HA-based compound areprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 indicates the degree of fluorescein staining in the case ofhaving administered a preparation to the eye of a model animal once aday.

FIG. 2 indicates the degree of fluorescein staining in the case ofhaving administered a preparation to the eye of a model animal six timesa day.

FIG. 3 indicates changes in a subjective symptom (ocular discomfort) inthe case of having administered a preparation to the eye of a humanpatient.

FIG. 4 indicates changes in a subjective symptom (dryness) in the caseof having administered a preparation to the eye of a human patient.

FIG. 5 indicates changes in a subjective symptom (grittiness) in thecase of having administered a preparation to the eye of a human patient.

FIG. 6 indicates changes in the worst of various subjective symptoms inthe case of having administered a preparation to the eye of a humanpatient.

FIG. 7 indicates changes in the average value of all subjective symptomsin the case of having administered a preparation to the eye of a humanpatient.

FIG. 8 indicates changes in the average value of subjective symptoms(ocular discomfort and dryness) in the case of having administered apreparation to the eye of a human patient.

FIG. 9 indicates the degree of fluorescein staining in the case ofhaving administered a preparation to the eye of a human patient.

MODE FOR CARRYING OUT THE INVENTION

In the present description, the term “step” not only refers to anindependent step, but also includes a step that cannot be clearlydistinguished from another step provided the intended objective of thatstep is achieved. In addition, unless specifically indicated otherwise,the content of each component of a composition refers to the totalamount of a plurality of those substances present in the composition inthe case a plurality of types of the substances are present for eachcomponent in the composition.

The following provides an explanation of embodiments of the presentinvention.

(1) Pharmaceutical Preparation of Present Invention

The pharmaceutical preparation of the present invention is an agentand/or pharmaceutical for improving human ocular subjective symptomsthat contains HA having an aminoalkyl cinnamate covalently bondedthereto (hereinafter referred to as the “present compound”).

(1-1) Present Compound

The present compound that is an active ingredient of the pharmaceuticalpreparation of the present invention is a compound in which anaminoalkyl cinnamate and HA are covalently bonded (hyaluronic acidderivative). Examples of the “aminoalkyl cinnamate” include aminoethylcinnamate and aminopropyl cinnamate. Among these, 2-aminoethyl cinnamateand 3-aminopropyl cinnamate are preferable, while 3-aminopropylcinnamate is particularly preferable. In the present description, andincluding the following explanation, it should be readily understoodthat the term “aminoalkyl cinnamate” includes and can be interchangedwith these specific and/or preferable cinnamic acid esters.

There are no particular limitations on the HA having this “aminoalkylcinnamate” covalently bonded thereto as far as it is a glycosaminoglycanwhich contains disaccharide units comprising N-acetyl-D-glucosamineattached to D-glucuronic acid in β1,3-linkage and the disaccharide unitsare connected each other repeatedly by β1,4-linkage. In addition, it maybe in a free state without forming a salt or may be in the form of apharmaceutically acceptable salt.

Examples of pharmaceutically acceptable salts of HA include alkalinemetal ion salts such as sodium salts or potassium salts, alkaline earthmetal salts such as magnesium salts or calcium salts, salts of inorganicbases such as ammonium salts, and salts of organic bases such asdiethanolamine, dicyclohexylamine or amino acids. The HA salt ispreferably a salt formed with an alkaline metal ion and particularly asalt formed with a sodium ion.

The HA may be any of that derived from a natural substance obtained byextracting from a portion of a living organism such as cockscomb,umbilical cord, cartilage or skin, that which has been chemicallysynthesized, and that which has been produced by microbial culturing orgenetic engineering techniques. Furthermore, since the present compoundis administered to a living organism, in addition to the presentcompound per se, the HA serving as the raw material thereof ispreferably also of high purity and substantially free of contaminantsfor which the presence thereof is not acceptable for pharmaceutical use.

There are no particular limitations on the weight-average molecularweight of the HA, and may be, for example, 10,000 to 5,000,000. Theweight-average molecular weight of the HA is preferably 200,000 to3,000,000 and more preferably 500,000 to 2,500,000. Furthermore, theweight-average molecular weight of the HA can be measured by thelimiting viscosity method.

The present compound can be produced by covalently bonding this HA withan aminoalkyl cinnamate. There are no particular limitations on the modeof this covalent bonding, and preferably the amino group of theaminoalkyl cinnamate is linked with the carboxyl group of HA through anamide linkage. The following provides an explanation using the presentcompound in which the covalent bond consists of an amide linkage as anexample.

In the present compound, all of the carboxyl groups of HA are notnecessarily required to form an amide linkage with the aminoalkylcinnamate, but rather only a portion of these groups may form an amidelinkage. In the subsequent descriptions, the ratio of all carboxylgroups present in HA that form an amide linkage is referred to as the“degree of substitution” (DS). DS is calculated from the ratio (%) ofthe number of aminoalkyl cinnamate residues introduced per constituentdisaccharide unit of HA. For example, the DS of an HA derivative inwhich one aminoalkyl cinnamate residue is introduced per constituentdisaccharide unit is 100%, while the DS of an HA derivative in which oneaminoalkyl cinnamate residue is introduced per 200 sugars (100constituent disaccharide units) is 1%.

The value of DS in the present compound is preferably 3-30%, morepreferably 10-20% and even more preferably 12-18%.

The present compound can be produced by linking an amino group derivedfrom an aminoalkanol that composes the aminoalkyl cinnamate (in themanner of, for example, an aminoethanol (such as 2-aminoethanol) or anaminopropanol (such as 3-aminopropanol)) with the carboxyl group of HA.

The aminoalkyl cinnamate is an ester compound in which the carboxylgroup of cinnamic acid and the hydroxyl group of the aminoalkanol arebonded through an ester linkage. Furthermore, the cinnamic acid thatcomposes the aminoalkyl cinnamate may be a substituted cinnamic acidhaving a substituent.

The present compound can be produced in compliance with the methoddescribed in, for example, Japanese Patent Application Laid-Open (JP-A)No. 2002-249501 or International Publication No. WO 2008/069348.

There are no particular limitations on the production method provided itallows the aminoalkyl cinnamate and HA to be chemically bonded throughan amide linkage. Examples of production methods include a method thatuses a water-soluble condensing agent such as a water-solublecarbodiimide (such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDCI.HCl) or 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate), a method that uses theaforementioned condensing agent and a condensation assistant such asN-hydroxysuccinimide (HOSu) or N-hydroxybenzotriazole (HOBt), a methodthat uses a condensing agent such as4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMT-MM), an active esterification method and an acid anhydride method.

The present compound may be prepared by preliminarily reacting cinnamicacid with an aminoalkanol (such as 3-aminopropanol, to apply similarlyhereinafter) to prepare an aminoalkyl cinnamate (such as 3-aminopropylcinnamate, to apply similarly hereinafter) followed by allowing theamino group of the aminoalkyl cinnamate to bond with the carboxyl groupof HA through an amide linkage, or by allowing the amino group of anaminoalkanol to bond with the carboxyl group of HA through an amidelinkage to prepare HA having an aminoalkanol introduced therein,followed by allowing the carboxyl group of cinnamic acid to bond with ahydroxyl group derived from the aminoalkanol present in theaforementioned HA having an aminoalkanol introduced therein through anester linkage.

(1-2) Formulation

The pharmaceutical preparation of the present invention can be producedby incorporating HA having an aminoalkyl cinnamate covalently bondedthereto and formulating into a preparation. Although there are noparticular limitations on the formulation method provided thepreparation can be made to be in the form of a preparation and/orpharmaceutical administered to the human eye, it is preferably in theform of a solution. This solution is preferably an ocular solution orophthalmic solution.

In addition, since the present compound demonstrates a remarkable and/orheterogeneous effect at a concentration of 0.3% (w/v), thepharmaceutical preparation of the present invention is preferably in theform of a solution and contains the present compound at a concentrationof 0.3% (w/v) as will be subsequently described.

In the formulation of such a preparation, the addition of apharmaceutically acceptable carrier as necessary, sterilization of thepreparation, and filling into a container suitable for administration tothe human eye can be suitably carried out by a person with ordinaryskill in the art.

To the pharmaceutical preparation of the present invention, it can beselected and added a buffering agent such as sodium phosphate, sodiumhydrogen phosphate, sodium dihydrogen phosphate, sodium acetate orε-aminocaproic acid, an isotonic agent such as sodium chloride,potassium chloride or concentrated glycerin, a surfactant such aspolyoxyethylene sorbitan monooleate, polyoxyl 40 stearate orpolyoxyethylene hydrogenated castor oil, or a preservative such asbenzalkonium chloride, as necessary.

In addition, although there are no particular limitations on the pH ofthe pharmaceutical preparation of the present invention within theacceptable range for the ophthalmic preparations, it is preferablywithin the range of pH 5 to 6.

(1-3) Method of Using Pharmaceutical Preparation of the PresentInvention

The pharmaceutical preparation of the present invention can be used byadministering to a human eye. Administration of the pharmaceuticalpreparation of the present invention to a human eye may be carried outby a method that is medically acceptable for administration to a humaneye, and there are no particular limitations thereon provided theeffects of the present invention can be demonstrated.

There are no particular limitations on the specific administrationmethod, and although the pharmaceutical preparation of the presentinvention may be suitably administered corresponding to the form of thepreparation, a preferable example thereof is instillation.

In addition, although there are no particular limitations on the numberof times or frequency of administration of the pharmaceuticalpreparation of the present invention, preferable examples thereofinclude administering 1 to 6 times per day, administering about 4 timesper day, administering everyday, continuously administering daily for 14days or longer and continuously administering daily for 28 days orlonger, while a combination thereof, (such as administering about 4times per day every day, continuously administering about 4 times perday for 14 days or more, or continuously administering about 4 times perday for 28 days or more) is more preferable. In addition, examples ofthe daily dosage include administration of 1, 2 or 3 drops peradministration.

Human ocular subjective symptoms can be improved by administering thepharmaceutical preparation of the present invention to a human eye.There are no particular limitations on the person targeted foradministration of the pharmaceutical preparation of the presentinvention provided he or she is a person exhibiting ocular subjectivesymptoms. In addition, there are no particular limitations on thepurpose of administration provided it is for improving human ocularsubjective symptoms.

There are no particular limitations on the “ocular subjective symptoms”described in the present description, and examples thereof includeocular discomfort (not including discomfort such as physical irritationcaused by administration of a preparation to the eye per se, butincluding the discomfort normally felt by dry eye patients), dryness(feeling that the eyes are dry), burning (feeling that the eyes are hotand smarting), feeling of a foreign body (strange feeling that somethingis contacting the eye during blinking or eyes feeling gritty),grittiness (feeling that the surface of the eye is not smooth as if sandhad entered the eyes), stinging (painful sensation as if pricked with aneedle), bleariness, itchiness, sensitivity to light and blurry vision,and the pharmaceutical preparation of the present invention can beapplied to one or two or more of these symptoms.

Since the pharmaceutical preparation of the present invention isparticularly effective for one or two or more subjective symptomsselected from the group consisting of ocular discomfort, dryness,burning, grittiness and stinging, among these, for one or two or moresubjective symptoms selected from the group consisting of oculardiscomfort, dryness, burning and grittiness, and especially for one ortwo or more subjective symptoms selected from the group consisting ofocular discomfort, dryness and grittiness, the pharmaceuticalpreparation of the present invention is preferably administered topersons having these symptoms or is preferably administered for thepurpose of improving these symptoms.

Since the pharmaceutical preparation of the present inventiondemonstrates effects that improve subjective symptoms, in particularimmediately after the start of administration, and is effective forimproving subjective symptoms at an early stage after the start ofadministration, it is preferably used for the purpose of improvingsubjective symptoms at an early stage after the start of administration.In the present description, “early stage after the start ofadministration” refers to a period of several weeks after the start ofadministration, preferably refers to a period up to and including aboutday 13, and more preferably refers to a period up to and including aboutday 6, when defining the day on which administration is started as day0.

In addition, in the present description, “immediately after the start ofadministration” refers to the day following the start of administration.

Among human ocular subjective symptoms, ocular discomfort and drynessare the most frequently indicated as chief complaints of dry eyepatients, accounting for roughly 80% of all complaints. As a result ofimproving these subjective symptoms at an early stage after the start ofadministration, or immediately after the start of administration inparticular, dry eye patients are encouraged to continue administrationof their own volition, making it possible to reliably demonstrateexpected therapeutic effects. As is indicated in the examples to besubsequently described, since the pharmaceutical preparation of thepresent invention demonstrated efficacy from immediately after the startof administration and at an early stage after the start ofadministration in analyses of the average values of scores used toevaluate these subjective symptoms, the pharmaceutical preparation ofthe present invention can be expected to promote voluntary continuationof administration by the patient per se as well as demonstratetherapeutic effects.

In addition, since the pharmaceutical preparation of the presentinvention is effective for improving the aforementioned subjectivesymptoms particularly in persons in which these symptoms are feltespecially strongly (persons with severe subjective symptoms such aspersons exhibiting an average score of 2.5 or higher for the worstsubjective symptom), the pharmaceutical preparation of the presentinvention is administered to such persons for that purpose. As explainedin the examples to be subsequently described, the “score” referred tohere indicates a score of 0 (no subjective symptoms) to 5 (worstsubjective symptoms) used to evaluate each of the aforementionedsymptoms in accordance with that perceived by the person administeredthe pharmaceutical preparation of the present invention. Among scoresgiven for each of the subjective symptoms, those persons having anaverage of 2.5 or higher for the highest score are considered to be“persons exhibiting an average score of 2.5 or higher for the worstsubjective symptom”. In addition, a person administered thepharmaceutical preparation of the present invention is preferably aperson with dry eye (in the present specification, dry eye has samemeaning with dry eye disease and dry eye syndrome).

Since the pharmaceutical preparation of the present invention isparticularly effective for improving subjective symptoms before bedtimeat which time subjective symptoms are thought to appear with thegreatest severity as a result of having used the eyes throughout theday, the pharmaceutical preparation of the present invention can be usedfor the purpose of improving subjective symptoms before bedtime in thismanner.

The term “improvement” of subjective symptoms in the present descriptionrefers to a decrease in subjective symptoms. Improvement of subjectivesymptoms can be evaluated quantitatively by a decrease in the subjectivesymptoms score.

(2) Method of Present Invention

The method of the present invention is a method for improving humanocular subjective symptoms that comprises a step for administering thepresent compound (which naturally includes that in the form of thepharmaceutical preparation of the present invention) to the eye of ahuman. The method of the present invention can be carried out in thesame manner as that in accordance with the explanation described in theaforementioned section (1) entitled “Pharmaceutical Preparation ofPresent Invention”. Thus, for example, administration of the presentcompound preferably being in the form of a 0.3% (w/v) solution and theperson receiving administration preferably being a person with dry eyeare the same as previously described.

The present invention includes the use of the present compound forimproving human ocular subjective symptoms and the use of thepharmaceutical preparation of the present invention for improving humanocular subjective symptoms.

EXAMPLES

The following provides an explanation of the present invention throughexamples thereof. However, the technical scope of the present inventionis not limited thereby.

[Example 1] Preparation of HA Having Aminoalkyl Cinnamate CovalentlyBonded Thereto

HA having an aminopropyl cinnamate covalently bonded thereto wasprepared in compliance with the method described in Example 2 of JP-A2002-249501 using HA having a weight-average molecular weight of 880,000(as measured according to the limiting viscosity method). In thefollowing descriptions, this “HA having an aminopropyl cinnamatecovalently bonded thereto” is abbreviated as “HA-3APC”. As a result ofanalyzing the resulting HA-3APC according to the method described in theexamples of JP-A 2002-249501, the degree of substitution of theaminopropyl cinnamate per HA repeating disaccharide unit was 15.3%.

[Example 2] Preparation of Ophthalmic Solution 1

After adding phosphate-buffered saline (PBS) to the HA-3APC (testsubstance) prepared in Example 1 and preparing solutions containingHA-3APC at 0.5 w/v %, 0.3 w/v % and 0.1 w/v %, the resulting solutionswere subjected to filtration sterilization using a 0.22 μm filter toobtain ophthalmic solutions. These ophthalmic solutions are referred toas the 0.5% test compound solution, 0.3% test compound solution and 0.1%test compound solution, respectively.

[Reference Example 1] Test Using Animal Dry Eye Disease Model

(1) Preparation of Model Animals

Seven-week-old SD male rats (SPF) were mildly anesthetized with diethylether following by shaving hair from the cheeks on both the left andright sides under isoflurane inhalation anesthesia.

After disinfecting the shaved areas with 70% ethanol solution, incisionswere made at locations about 7 mm below the ears that extended about 7mm in the longitudinal direction followed by excision of theextraorbital lacrimal glands of both eyes. Next, an antimicrobial agent(Tarivid™ ophthalmic ointment) was applied to the incision wound, andafter suturing the wound, the sutured wound was disinfected with 10%povidone-iodine solution.

Two months after excision of the lacrimal glands, the corneal epitheliaof both eyes were subjected to fluorescein staining using Fluores™ TestPaper (Showa Yakuhin Kako Co., Ltd.) under isoflurane inhalationanesthesia. As a result, defective areas (damaged areas) of the cornealepithelia were fluorescein-stained.

The entire cornea was macroscopically divided from above into threesections while illuminated with a slit lamp (SL-D7, Topcon Corp.) andthe degree of damage to the corneal epithelia was evaluated based on thefollowing criteria for each section (maximum score of 9 per eye). Thescores for each animal were indicated as the average of both eyes. Inaddition, the entire cornea of each animal scored was photographed usinga digital photography unit.

(Criteria)

0 points: Absence of stained dots

1 point: Sparse (fluorescein-stained dots are separated from each other)

2 points: Moderate (intermediate to sparse and dense)

3 points: Dense (nearly all fluorescein-stained dots are adjacent toeach other)

(2) Grouping

The model animals prepared in the manner described above were dividedinto the 9 groups shown in Table 1 after scoring so that the averagescores were the same among the groups.

TABLE 1 Number of Dosage Dosing Administered Animals per admin-frequency Group Substance (Eyes) istration (times/day) Control group PBS5 (10) 5 μL 1 time (once per day) Control group PBS 5 (10) 5 μL 6 times(6 times per day) 0.1% group 0.1% test sub- 5 (10) 5 μL 1 time (once perday) stance solution 0.3% group 0.3% test sub- 5 (10) 5 μL 1 time (onceper day) stance solution 0.5% group 0.5% test sub- 5 (10) 5 μL 1 time(once per day) stance solution 0.1% group 0.1% test sub- 5 (10) 5 μL 6times (6 times per day) stance solution 0.3% group 0.3% test sub- 5 (10)5 μL 6 times (6 times per day) stance solution 0.5% group 0.5% test sub-5 (10) 5 μL 6 times (6 times per day) stance solution Normal group None3 (6) None None

(3) Test Method

The test substance was administered to each group in accordance withTable 1. The test substance was administered by instillation using acontinuous dispenser (Multipette Plus, Eppendorf Co.). Furthermore,those groups that were dosed 6 times per day were dosed a total of 6times at 1.5 hour intervals, those groups dosed once per day were dosedonce per day at the time of the initial dosing of the groups dosed 6times per day, and this was continued for 21 days (3 weeks). The degreeof damage to corneal epithelia was evaluated immediately after the startof administration (day 0), on day 3 and at the days after 1 week, 2weeks and 3 weeks. Damage was evaluated in accordance with theaforementioned criteria and scored under blind conditions.

The results for each group were indicated as the mean±standard error.The scores for each group dosed with a test substance solution at eachevaluation time were confirmed for dose response according to theShirley-Williams test and Jonckheere-Terpstra test. A level ofsignificance of less than 5% was considered to be significant.

(4) Test Results

The results are shown in FIGS. 1 and 2. According to the results of thedose-response relationship test as determined according to theShirley-Williams test, among those groups dosed once per day (FIG. 1),significant differences were observed versus the control group (once perday) for the group dosed at 0.5% at all evaluation times after day 3 andfor the groups dosed at 0.1% and 0.3% at the days after the 1 week and 3weeks. In addition, among those groups dosed 6 times per day (FIG. 2),significant differences were observed versus the control group (6 timesper day) for all groups dosed with the test substance at all evaluationtimes after day 3.

In addition, according to the results of the dose-response trend test asdetermined according to the Joncldieere-Terpstra test, among thosegroups dosed once per day (FIG. 1), the Jonckherre statistic wassignificant on day 3 and at the days after the 2 week and 3 weeks, andamong those groups dosed 6 times per day (FIG. 2), the Jonckherrestatistic was significant at all evaluation times.

Since dosing once per day and dosing 6 times per day were significantfor both tests at the time of the final evaluation at the day after the3 weeks, a dose-response relationship was indicated regardless of theadministration frequency per day.

[Example 3] Preparation of Ophthalmic Solution 2

A base (consisting of sodium chloride and potassium chloride as isotonicagents, sodium hydrogen phosphate and sodium dihydrogen phosphate asbuffering agents, disodium edetate as stabilizer, and 0.003%benzalkonium chloride as preservative) was added to the HA-3APC preparedin Example 1, and after adjusting the pH to 5.0 to 6.0 to prepare 0.5w/v % and 3.0 w/v % HA-3APC solutions, the solutions were subjected tofiltration sterilization with a 0.22 μm filter to obtain ophthalmicsolutions. These solutions are referred to as a 0.5% ophthalmic solutionand 0.3% ophthalmic solution, respectively. In addition, a base solutionnot containing HA-3APC was prepared for use as a placebo.

[Example 4] Clinical Study in Dry Eye Patients Using CAE™ Model

(1) Subjects

Dry eye patients who satisfied all of the following conditions of A) toC) were enrolled in the study:

A) Age 18 or older,

B) Provided written informed consent, and

C) History of dry eye in both eyes.

However, dry eye patients applicable to any of the following a) to d)were excluded from the study:

a) Wear contact lenses,

b) Have an uncontrollable systemic disease,

c) Have an uncontrollable mental disease, drug addiction or alcoholaddiction, and

d) Lactating or pregnant women and women planning to become pregnant.

(2) Study Method

A joint multicenter, randomized, double-blind comparative study wasconducted using a CAE™ (Controlled Adverse Environment) model (Ora,Inc., Andover, Mass., USA) (refer to George W. Ouster, et al., AnnAllergy Asthma Immunol, 2004; 93: 460-464), which has been establishedto evaluate the therapeutic effects of the treatments for dry eye in thefield of ophthalmology, in which the humidity kept low, temperature, airflow, lighting conditions and visual tasking were controlled.

First, as an introduction to the study, the subjects dropped a placebointo both eyes at 1 drop per administration 4 times per day (morning,noon, afternoon and before bedtime), and this was continued for 2 weeksas a run-in period. The subjects were instructed to score ocularsubjective symptoms (ocular discomfort, dryness, burning, grittiness andstinging) based on their own perception of those symptoms and recordthem in a daily diary at bedtime (prior to instillation before bedtime)every day during the run-in period. The scores ranged from 0 (absence ofsubjective symptoms) to 5 (most severe (worst) subjective symptoms).

(3) Administration of Ophthalmic Solutions

Next, the subjects dropped in the 0.5% ophthalmic solution or 0.3%ophthalmic solution prepared in Example 3 or the placebo in both eyes at1 drop per administration 4 times per day (morning, noon, afternoon andbefore bedtime), and this was continued for 4 weeks. The subjects wereinstructed to score subjective symptoms and record them in a daily diaryat bedtime (prior to instillation before bedtime) every day during theinstillation period.

(4) Results

Subjects who strongly perceived subjective symptoms (subjects having anaverage score of 2.5 or higher for the most severe (highest) subjectivesymptom score (worst symptom score)) immediately before startingadministration of ophthalmic solution (period of 7 days to 1 day beforethe start of administration of ophthalmic solution) were evaluated, andtheir time-based changes during the administration period were graphed.

Differences from the average subjective symptom score immediately beforeinstillation performed on day 0 (period of 7 days to 1 day before thestart of administration of ophthalmic solution) in each dose group(baseline) were used for evaluation. In addition, the average score foreach subjective symptom in each dose group at each evaluation time wasused for this evaluation. The results are shown in FIGS. 3 to 8.

Furthermore, since these results indicate results obtained at bedtime(prior to instillation before bedtime) when eye fatigue had accumulatedfollowing continuous use of the eyes throughout the day, they areconsidered to represent the strongest appearance of subjective symptomswhile also having a considerable effect on sleep. Namely, the followingresults were obtained by evaluating those patients presenting withsevere subjective symptoms at a time of the day when the symptoms arethought to appear most intensely.

Furthermore, the number of subjects in the 0.5% ophthalmic solution,0.3% ophthalmic solution and placebo groups were 23, 26 and 26,respectively.

(4-1) Ocular Discomfort

Results for ocular discomfort are shown in FIG. 3. As a result ofanalyzing these results, statistically significant decreases insubjective symptoms versus the placebo group were observed in the 0.3%ophthalmic solution group during the time periods indicated below.

-   -   Day 0 to Day 6 (P=0.0219, t-test; P=0.0249, ANCOVA)    -   Day 0 to Day 13 (P=0.0143, t-test; P=0.0210, ANCOVA)    -   Day 0 to Day 20 (P=0.0387, t-test)    -   Day 7 to Day 13 (P=0.0233, t-test; P=0.0407, ANCOVA)

On the other hand, a statistically significant difference versus theplacebo group was observed in the 0.5% ophthalmic solution group onlyduring the period from Day 21 to Day 27 (P=0.0306, test method: t-test).

In this manner, these results surprisingly showed that the 0.3%ophthalmic solution demonstrated an ameliorative effect on human ocularsubjective symptoms (ocular discomfort) at an early stage after thestart of administration of ophthalmic solution (Day 0 to Day 20), andthat the effect was superior to that of the 0.5% ophthalmic solution.

(4-2) Dryness

The results for dryness are shown in FIG. 4. As a result of analyzingthese results, statistically significant decreases in subjectivesymptoms versus the placebo group were observed in the 0.3% ophthalmicsolution group during the time periods indicated below.

-   -   Day 0 to Day 6 (P=0.0480, t-test)    -   Day 0 to Day 13 (P=0.0173, t-test; P=0.0259, ANCOVA)    -   Day 0 to Day 20 (P=0.0466, t-test)    -   Day 7 to Day 13 (P=0.0140, t-test; P=0.0240, ANCOVA)    -   Day 0 to Day 27 (P=0.0365, Wilcoxon rank sum)

On the other hand, statistically significant differences were notobserved in the 0.5% ophthalmic solution group.

In this manner, these results surprisingly showed that the 0.3%ophthalmic solution demonstrated an ameliorative effect on human ocularsubjective symptoms (dryness) at an early stage after the start ofadministration of ophthalmic solution (Day 0 to Day 27), and that theeffect was superior to that of the 0.5% ophthalmic solution.

(4-3) Grittiness

The results for grittiness are shown in FIG. 5. As a result of analyzingthese results, a statistically significant decrease in subjectivesymptoms versus the placebo group was observed in the 0.3% ophthalmicsolution group during the time period from day 0 to day 13 (P=0.0286,Wilcoxon rank sum).

On the other hand, statistically significant differences were notobserved in the 0.5% ophthalmic solution group.

In this manner, these results surprisingly showed that the 0.3%ophthalmic solution demonstrated an ameliorative effect on human ocularsubjective symptoms (grittiness) at an early stage after the start ofadministration of ophthalmic solution (Day 0 to Day 13), and that theeffect was superior to that of the 0.5% ophthalmic solution.

(4-4) Worst Subjective Symptom Score

FIG. 6 indicates the results for the subjective symptom perceived byeach patient (ocular discomfort, burning, dryness, grittiness andstinging) to be the worst subjective symptom (highest symptom score).

As a result of analyzing these results, statistically significantdecreases in subjective symptoms versus the placebo group were observedin the 0.3% ophthalmic solution group during the time periods indicatedbelow.

-   -   Day 0 to Day 13 (P=0.0232, t-test; P=0.0324, ANCOVA)    -   Day 7 to Day 13 (P=0.0204, t-test; P=0.0308, ANCOVA)

On the other hand, statistically significant differences were notobserved in the 0.5% ophthalmic solution group.

In this manner, these results surprisingly showed that the 0.3%ophthalmic solution demonstrated an ameliorative effect on the worsthuman ocular subjective symptoms at an early stage after the start ofadministration of ophthalmic solution (Day 0 to Day 13), and that theeffect was superior to that of the 0.5% ophthalmic solution.

(4-5) Average of Subjective Symptom Score

FIG. 7 indicates the results of analyzing the average score of eachpatient for each subjective symptom (ocular discomfort, dryness,burning, grittiness and stinging).

Statistically significant decreases in subjective symptoms versus theplacebo group were observed in the 0.3% ophthalmic solution group duringthe time periods indicated below.

-   -   Day 0 to Day 6 (P=0.0481, t-test)    -   Day 0 to Day 13 (P=0.0404, t-test; P=0.0422, ANCOVA)    -   Day 7 to Day 13 (P=0.0148, Wilcoxon rank sum)

On the other hand, statistically significant differences were notobserved in the 0.5% ophthalmic solution group.

In this manner, these results surprisingly showed that the 0.3%ophthalmic solution demonstrated an ameliorative effect across theentire spectrum of human ocular subjective symptoms at an early stageafter the start of administration of ophthalmic solution (Day 0 to Day6), and that the effect was superior to that of the 0.5% ophthalmicsolution.

(4-6) Average of Subjective Symptom Score (Ocular Discomfort, Dryness)

FIG. 8 indicates the results of analyzing the average score of eachpatient for each subjective symptom (ocular discomfort and dryness).

Statistically significant decreases in these subjective symptoms versusthe placebo group were observed in the 0.3% ophthalmic solution groupduring the time periods indicated below.

-   -   Day 0 to Day 13 (P=0.0111, t-test; P=0.0080, Wilcoxon rank sum;        P=0.0138, ANCOVA)    -   Days 1, 4, 5, 8, 9 and 10 (P=0.0235, 0.0066, 0.0046, 0.0018,        0.0313 and 0.0145, respectively, t-test)

On the other hand, statistically significant differences were notobserved in the 0.5% ophthalmic solution group.

In this manner, these results surprisingly showed that the 0.3%ophthalmic solution demonstrated an ameliorative effect on the humanocular subjective symptoms of ocular discomfort and dryness, for whichcomplaints from patients are the most numerous, at an early stage afterthe start of administration of ophthalmic solution (and surprisingly, onthe day following the start of administration of ophthalmic solution),and that the effect was superior to that of the 0.5% ophthalmicsolution.

(5) Changes in Fluorescein Staining Scores Before and After CAE™

Changes in fluorescein staining (staining of damaged areas of cornealand conjunctival epithelia) scores for the entire corneal and entireconjunctiva before and after CAE™ were evaluated for each subject on day28. The results are shown in FIG. 9.

As a result of analyzing the results, statistically significantdecreases in staining scores versus the placebo group were observedthroughout the entire cornea and conjunctiva in the 0.3% ophthalmicsolution group.

On the other hand, there were no statistically significant differencesin the 0.5% ophthalmic solution group for either the entire cornea orentire conjunctiva.

In this manner, these results surprisingly showed that the 0.3%ophthalmic solution demonstrated an effect that brought about a decreasein the occurrence of damage to corneal and conjunctival epithelia inhuman eyes, and that the effect was superior to that of the 0.5%ophthalmic solution.

(6) Evaluation of Safety

There were no statistically significant differences observed among the0.3% ophthalmic solution group, 0.5% ophthalmic solution group andplacebo group with respect to the incidence of systemic adverse events,the incidence of ocular adverse events or the incidence of adverse sideeffects, and there were no events observed that warranted concern overthe safety of HA-3APC.

Disclosures of Japanese Patent Application No. 2001-385072 (filing date:Dec. 18, 2001), Japanese Patent Application No. 2008-519554 (filingdate: Oct. 12, 2006), and Japanese Patent Application No. 2014-263710(filing date: Dec. 26, 2014) are incorporated in the presencedescription in their entirety by reference.

All publications, patent applications and technical standards describedin the present description are incorporated herein by reference to thesame extent as if each publication, patent application or technicalstandard was specifically and individually indicated to be incorporatedby reference.

INDUSTRIAL APPLICABILITY

The pharmaceutical preparation of the present invention can be appliedindustrially as a preparation for improving human ocular subjectivesymptoms, while the method of the present invention can be appliedindustrially as a method for improving human ocular subjective symptomsthat uses an HA-based compound.

The invention claimed is:
 1. An ophthalmic solution, comprising ahyaluronic acid having an aminoalkyl cinnamate covalently bonded theretoor a pharmaceutically acceptable salt thereof, wherein said ophthalmicsolution is a pharmaceutical preparation containing said hyaluronic acidhaving an aminoalkyl cinnamate covalently bonded thereto or saidpharmaceutically acceptable salt in a concentration of 0.3% (w/v) as anactive ingredient, and wherein a pH of said ophthalmic solution iswithin the range of not less than 5 and not more than
 6. 2. Theophthalmic solution according to claim 1, which has been sterilized. 3.The ophthalmic solution according to claim 1, which is contained in acontainer suitable for administering said ophthalmic solution to a humaneye.